QUAFIT: A Novel Method for the Quaternary Structure Determination from Small-Angle Scattering Data

The new QUAFIT method for determining the quaternary structure of biological macromolecular assemblies by analyzing x-ray or neutron small-angle scattering data is presented. The method is based on the idea that asymmetric monomers, formed by rigid domains of known atomic structure possibly connected by flexible linkers of known sequence, are assembled according to a point-group symmetry combined with a screw axis. Scattering amplitudes of domains and linkers are determined by means of a spherical harmonics expansion and combined to get the form factor of the assembly. To avoid any overlap among domains, the contact distance between two asymmetric domains is determined as a function of their orientation by a new algorithm, based on Stone's Invariants expansion. To account for continuity and compactness of the whole assembly, an anisotropic Lennard-Jones potential among domains, written in terms of the contact distances, is included in the merit function. QUAFIT allows for the simultaneous presence of oligomerization intermediates as well as of monomers distributed over multiple conformations. QUAFIT has been tested by studying the structure of a high molecular weight protein, the hemocyanin from Octopus vulgaris, under solution conditions that stabilize the decameric form or induce dissociation into monomers, respectively. Results are in very good agreement with the structural model derived from electron microscopy observations.     

The role of copper and quaternary structure on the conformational properties of Octopus vulgaris hemocyanin

Some structural properties of Octopus vulgaris hemocyanin have been investigated by fluorescence spectroscopy. The three-dimensional structure of Octopus hemocyanin is remarkably tight, resulting in a deep burial of almost all the tryptophyl residues of the protein. The hemocyanin conformation has been studied in the two main aggregation states (11 S, 50 S) of the protein, and with respect to the presence or absence of copper in the active site. Upon changing the pH of the solution, Octopus hemocyanin in the 50 S aggregation state can assume at least three different conformations. During the transition between each conformation the fluorescence quantum yield changes, but the environment of tryptophans does not change. Dissociation of the protein from 50 S to 11 S strongly enhances its susceptibility toward denaturating agents such as pH or temperature, and modifies the effects of fluorescence quenchers such as acrylamide. Moreover, these effects are more pronounced when copper is removed from the active site. A comparative analysis of the results shows that the subunit-subunit interactions exerted within the 50 S species are more important in the maintenance of the conformational stability than the copper ions present in the active sites. This behavior can be accounted for by the large amount of Ca(II) ions linked to 50 S hemocyanin.     

Type-3 copper proteins as biocompatible and reusable oxygen sensors

Fluorescently labeled type-3 copper proteins have been proposed previously as solution oxygen sensors by using a FRET mechanism. Herein, we describe how this principle can be adapted to sense O₂ by means of proteins immobilized in optically transparent silica matrices. Specifically, the protein, hemocyanin from Octopus vulgaris N-terminally labeled with Cy5, is immobilized in two different kinds of optically transparent silica matrices, which appear to be a promising platform for enzyme encapsulation. The presented results provide proof of principle that fluorescently labeled proteins immobilized in a silica matrix can be implemented in a reusable, biocompatible and stable oxygen measuring device that might lead to new developments in the field of optical biosensing.     

The stabilizing influence of divalent ions and Na+ on the di-decameric structure of Yoldia limatula hemocyanin

The stabilizing influence of Ca²⁺, Mg²⁺, Ba²⁺ and Na⁺ on the di-decameric structure of the hemocyanin of the bivalve, Yoldia limatula has been investigated by light-scattering molecular weight measurements and by analytical ultracentrifugation. The molecular weight (M_w) data, examined as a function of decreasing divalent ion and sodium ion concentrations at pH 8.0 and at a constant hemocyanin concentration of 0.10 g·l⁻¹, show biphasic transition profiles, with a sharp initial decline in M_w as the concentration of the stabilizing cations is reduced. The analysis of the molecular weight data is best described in terms of the four-species, di-decamer-decamer-dimer-monomer scheme of association-dissociation equilibria. About 25 to 35 bound divalent ions and about 10 bound Na⁺ ions per half-molecule or decamer are required in order to account for the initial step of the observed transitions. The subsequent transitions representing the decamer to dimer and the dimer to nonomer steps of the reaction account for the additional binding of three to four and two to four cations per dimer and per monomer, respectively. The relatively large number of divalent ions per decamer suggests strong ionic stabilization of the decamer to decamer contacts within the parent di-decameric assembly of Yoldia hemocyanin. This is consistent with earlier observations showing relatively few hydrophobic groups at the decamer to decamer contact areas.     

Structural and Thermodynamic Properties of Septin 3 Investigated by Small-Angle X-Ray Scattering

Septins comprise a family of proteins involved in a variety of cellular processes and related to several human pathologies. They are constituted by three structural domains: the N- and C-terminal domains, highly variable in length and composition, and the central domain, involved in the guanine nucleotide (GTP) binding. Thirteen different human septins are known to form heterogeneous complexes or homofilaments, which are stabilized by specific interactions between the different interfaces present in the domains. In this work, we have investigated by in-solution small-angle x-ray scattering the structural and thermodynamic properties of a human septin 3 construct, SEPT3-GC, which contains both of both interfaces (G and NC) responsible for septin-septin interactions. In order to shed light on the role of these interactions, small-angle x-ray scattering measurements were performed in a wide range of temperatures, from 2 up to 56°C, both with and without a nonhydrolysable form of GTP (GTP_γS). The acquired data show a temperature-dependent coexistence of monomers, dimers, and higher-order aggregates that were analyzed using a global fitting approach, taking into account the crystallographic structure of the recently reported SEPT3 dimer, PDB:3SOP. As a result, the enthalpy, entropy, and heat capacity variations that control the dimer-monomer dissociation equilibrium in solution were derived and GTPγS was detected to increase the enthalpic stability of the dimeric species. Moreover, a temperature increase was observed to induce dissociation of SEPT3-GC dimers into monomers just preceding their reassembling into amyloid aggregates, as revealed by the Thioflavin-T fluorescence assays.     

Molluscan mega-hemocyanin: an ancient oxygen carrier tuned by a ~550 kDa polypeptide

Background

The allosteric respiratory protein hemocyanin occurs in gastropods as tubular di-, tri- and multimers of a 35 × 18 nm, ring-like decamer with a collar complex at one opening. The decamer comprises five subunit dimers. The subunit, a 400 kDa polypeptide, is a concatenation of eight paralogous functional units. Their exact topology within the quaternary structure has recently been solved by 3D electron microscopy, providing a molecular model of an entire didecamer (two conjoined decamers). Here we study keyhole limpet hemocyanin (KLH2) tridecamers to unravel the exact association mode of the third decamer. Moreover, we introduce and describe a more complex type of hemocyanin tridecamer discovered in fresh/brackish-water cerithioid snails (Leptoxis, Melanoides, Terebralia).

Results

The "typical" KLH2 tridecamer is partially hollow, whereas the cerithioid tridecamer is almost completely filled with material; it was therefore termed "mega-hemocyanin". In both types, the staggering angle between adjoining decamers is 36°. The cerithioid tridecamer comprises two typical decamers based on the canonical 400 kDa subunit, flanking a central "mega-decamer" composed of ten unique ~550 kDa subunits. The additional ~150 kDa per subunit substantially enlarge the internal collar complex. Preliminary oxygen binding measurements indicate a moderate hemocyanin oxygen affinity in Leptoxis (p50 ~9 mmHg), and a very high affinity in Melanoides (~3 mmHg) and Terebralia (~2 mmHg). Species-specific and individual variation in the proportions of the two subunit types was also observed, leading to differences in the oligomeric states found in the hemolymph.

Conclusions

In cerithioid hemocyanin tridecamers ("mega-hemocyanin") the collar complex of the central decamer is substantially enlarged and modified. The preliminary O₂ binding curves indicate that there are species-specific functional differences in the cerithioid mega-hemocyanins which might reflect different physiological tolerances of these gill-breathing animals. The observed differential expression of the two subunit types of mega-hemocyanin might allow individual respiratory acclimatization. We hypothesize that mega-hemocyanin is a key character supporting the adaptive radiation and invasive capacity of cerithioid snails.     

Orienting rigid and flexible biological assemblies in ferrofluids for small-angle neutron scattering studies

Small-angle scattering from macromolecules in solution is widely used to study their structures, but the information content is limited because the molecules are generally randomly oriented and hence the data are spherically averaged. The use of oriented rodlike structures for scattering, as in fiber diffraction, greatly increases the amount of structural detail that can be obtained. A new technique using a ferromagnetic fluid has been developed to align elongated structures independent of their intrinsic magnetic properties. This technique is ideal for small-angle neutron scattering because the scattering from the ferrofluid particles can be reduced significantly by matching the neutron scattering length density of the particles to a D₂O solvent (“contrast matching”). The net result is scattering primarily from the ordered biological assembly in a solution environment that can be adjusted to physiological pH and ionic strength. Scattering results from ordered tobacco mosaic virus, tobacco rattle virus, and chromain fibers are presented.     

Co(II)-substituted Octopus vulgaris hemocyanin

Co(II)-substituted hemocyanin (Co(II)Hc) of the octopus, Octopus vulgaris, has been prepared by dialysis of apohemocyanin against Co(II·) ion and subsequent Chelex-treatment. The blue 50%-Co(II)Hc (half-apo Co(II)Hc), in which binuclear coppers are replaced in the hemocyanin by a single Co(II), exhibits two absorption maxima at 560 (?_Co=250) and 594 nm (?_Co=320 M^?1 cm^?1) and a shoulder near 610 nm, all of which are attributed to a dd transition of high spin Co(II) (S=3/2) with a tetrahedral geometry. The magnetic circular dichroism (MCD) spectrum in this region also suggests the existence of a tetrahedral Co(II) species in the protein. The visible absorption and MCD spectra of octopus 50%-Co(II)Hc are quite similar to those of squid 50%-Co(II)Hc described in the previous paper (S. Suzuki, J. Kino, M. Kimura, W. Mori and A. Nakahara, Inorg. Chim. Acta, 66, 41 (1982)). The formation of half-apo Co(II)Hc demonstrates that the binuclear copper sites in native octopus hemocyanin may differ from each other in coordination geometry, as in other molluscan hemocyanins, squid and snail hemocyanins. The coordination environment of the active-site Co(II) substituted for Cu in the octopus hemocyanin is the same as that of the corresponding active site of the squid hemocyanin.     

Effects of Macromolecular Crowding on the Structure of a Protein Complex: A Small-Angle Scattering Study of Superoxide Dismutase

Macromolecular crowding can alter the structure and function of biological macromolecules. We used small-angle scattering to measure the effects of macromolecular crowding on the size of a protein complex, SOD (superoxide dismutase). Crowding was induced using 400 MW PEG (polyethylene glycol),TEG (triethylene glycol), α-MG (methyl-α-glucoside), and TMAO (trimethylamine n-oxide). Parallel small-angle neutron scattering and small-angle x-ray scattering allowed us to unambiguously attribute apparent changes in radius of gyration to changes in the structure of SOD. For a 40% PEG solution, we find that the volume of SOD was reduced by 9%. Considering the osmotic pressure due to PEG, this deformation corresponds to a highly compressible structure. Small-angle x-ray scattering done in the presence of TEG suggests that for further deformation—beyond a 9% decrease in volume—the resistance to deformation may increase dramatically.     

Probing the Conformation of FhaC with Small-Angle Neutron Scattering and Molecular Modeling

Probing the solution structure of membrane proteins represents a formidable challenge, particularly when using small-angle scattering. Detergent molecules often present residual scattering contributions even at their match point in small-angle neutron scattering (SANS) measurements. Here, we studied the conformation of FhaC, the outer-membrane, β-barrel transporter of the Bordetella pertussis filamentous hemagglutinin adhesin. SANS measurements were performed on homogeneous solutions of FhaC solubilized in n-octyl-d17-βD-glucoside and on a variant devoid of the α helix H1, which critically obstructs the FhaC pore, in two solvent conditions corresponding to the match points of the protein and the detergent, respectively. Protein-bound detergent amounted to 142 ± 10 mol/mol as determined by analytical ultracentrifugation. By using molecular modeling and starting from three distinct conformations of FhaC and its variant embedded in lipid bilayers, we generated ensembles of protein-detergent arrangement models with 120–160 detergent molecules. The scattered curves were back-calculated for each model and compared with experimental data. Good fits were obtained for relatively compact, connected detergent belts, which occasionally displayed small detergent-free patches on the outer surface of the β barrel. The combination of SANS and modeling clearly enabled us to infer the solution structure of FhaC, with H1 inside the pore as in the crystal structure. We believe that our strategy of combining explicit atomic detergent modeling with SANS measurements has significant potential for structural studies of other detergent-solubilized membrane proteins.     

The Hemocyanin from a Living Fossil, the Cephalopod Nautilus pompilius: Protein Structure, Gene Organization, and Evolution

By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a ring-like decamer (M_r = ∼3.5 million), assembled from 10 identical copies of an ∼350-kDa polypeptide. This subunit in turn is substructured into seven sequential covalently linked functional units of ∼50 kDa each (FUs a–g). We have cloned and sequenced the cDNA encoding the complete polypeptide; it comprises 9198 bp and is subdivided into a 5′ UTR of 58 bp, a 3′ UTR of 365 bp, and an open reading frame for a signal peptide of 21 amino acids plus a polypeptide of 2903 amino acids (M_r = 335,881). According to sequence alignments, the seven FUs of Nautilus hemocyanin directly correspond to the seven FU types of the previously sequenced hemocyanin “OdH” from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed among the seven Nautilus hemocyanin FUs; the structural consequences of putatively attached glycans are discussed on the basis of the published X-ray structure for an Octopus dofleini and a Rapana thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to linker introns but shows two internal introns that differ in position from the three internal introns of the Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock. This reveals that the last common ancestor of Nautilus and Octopus lived 415 ± 24 million years ago, in close agreement with fossil records from the early Devonian. [Reviewing Editor: Dr. Axel Meyer] The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ619741.     

Nautilus pompilius hemocyanin: 9 A cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs. Nautilus pompilius (Cephalopoda) hemocyanin is a cylindrical decamer of a 350 kDa polypeptide subunit that in turn is a “pearl-chain” of seven different functional units (FU-a to FU-g). Each globular FU has a binuclear copper centre that reversibly binds one O₂ molecule, and the 70-FU decamer is a highly allosteric protein. Its primary structure and an 11 Å cryo-electron microscopy (cryo-EM) structure have recently been determined, and the crystal structures of two related FU types are available in the databanks. However, in molluscan hemocyanin, the precise subunit pathway within the decamer, the inter-FU interfaces, and the allosteric unit are still obscure, but this knowledge is crucial to understand assembly and allosterism of these proteins. Here we present the cryo-EM structure of Nautilus hemocyanin at 9.1 Å resolution (FSC_1/2-bit criterion), and its molecular model obtained by rigid-body fitting of the individual FUs. In this model we identified the subunit dimer, the subunit pathway, and 15 types of inter-FU interface. Four interface types correspond to the association mode of the two protomers in the published Octopus FU-g crystal. Other interfaces explain previously described morphological structures such as the fenestrated wall (which shows D5 symmetry), the three horizontal wall tiers, the major and minor grooves, the anchor structure and the internal collar (which unexpectedly has C5 symmetry). Moreover, the potential calcium/magnesium and N-glycan binding sites have emerged. Many interfaces have amino acid constellations that might transfer allosteric interaction between FUs. From their topologies we propose that the prime allosteric unit is the oblique segment between major and minor groove, consisting of seven FUs from two different subunits. Thus, the 9 Å structure of Nautilus hemocyanin provides fundamentally new insight into the architecture and function of molluscan hemocyanins.     

Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects

Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the R_P enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an S_P-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away.     

Effects of Heterogeneous Competitor Distribution and Ramet Aggregation on the Growth and Size Structure of a Clonal Plant

Spatially heterogeneous distribution of interspecific competitors and intraspecific aggregation of offspring ramets may affect the growth and size structure of clonal plant populations, but these have been rarely studied. We conducted a greenhouse experiment in which we grew a population of eight offspring ramets (plants) of the stoloniferous clonal plant Hydrocotyle vulgaris aggregately or segregately in two homogeneous treatments with or without a competing grass Festuca elata and a heterogeneous treatment with a patchy distribution of the grass. In patchy grass treatments, H. vulgaris produced markedly more biomass, ramets and stolons in open patches (without grasses) than in grass patches, but displayed lower size variations as measured by coefficient of variation of biomass, ramets and stolons among the eight plants. In open areas, H. vulgaris produced statistically the same amounts of biomass and even more stolons and showed higher size variations in patchy grass treatments than in open (no grass) treatments. In grass areas, H. vulgaris grew much worse and displayed higher size variations in patchy grass treatments than in full grass treatments. Ramet aggregation decreased the growth of H. vulgaris in open treatments and in both open and grass patches in patchy grass treatments, but had little effect in full grass treatments. Ramet aggregation had little effect on size variations. Therefore, heterogeneous distribution of competitors can affect the growth and size structure of clonal plant populations, and ramet aggregation may decrease population growth when they grow in open environments or heterogeneous environments with a patchy distribution of interspecific competitors.     

Small-angle X-ray scattering studies on the X-ray induced aggregation of malate synthase

Summary Malate synthase was investigated by the small-angle X-ray scattering technique in aqueous solution. Measurements extending for several hours revealed a continuous increase of the intensity in the innermost portion of the scattering curve. There is clear evidence that this increase was caused by an X-ray induced aggregation of enzyme particles during the performance of the small-angle X-ray scattering experiment. The monitoring of the aggregation process in situ by means of small-angle X-ray scattering led to a model of the way how the aggregation might proceed. The analysis of the scattering curves of malate synthase taken at various stages of aggregation established the retention of the thickness factor of the native enzyme and the occurrence of one and later on of two cross-section factors. The process of aggregation was also reflected by the increase of extension of the distance distribution function. According to these results, the first step of aggregation might be a linear side-by-side association of the oblate enzyme particles, a process which is followed by a twodimensional aggregation. An aggregation in the third dimension was not observed during the time covered by our experiment. The predominance of aggregation in only one or two dimensions was corroborated by comparison of appropriate theoretical scattering curves with the experimental curves. The theoretical scattering curves for this comparison were obtained by averaging over the properly weighted scattering curves calculated for various species of hypothetical aggregates. The time dependence of the apparent mean radius of gyration was used to compare the aggregation of enzyme samples that were irradiated under different experimental conditions. It turned out that by addition of dithiothreitol to the enzyme solutions as well as in the presence of the substrates (acetyl-CoA, glyoxylate) or of a substrate analogue (pyruvate) or of ethanol the rate of aggregation is reduced. Enzymic activity was found to decrease about exponentially with increasing X-ray dose. The presence of dithiothreitol or of the substrate glyoxylate or of the substrate analogue pyruvate protects the enzyme against X-ray induced inactivation. The substrate acetyl-CoA does not exhibit a comparable protective effect against inactivation. Measurements of enzymic activity and small-angle X-ray scattering on samples, which had been X-irradiated with a defined dose prior to the measurements, established two different series of efficiency for the protection of the enzyme against aggregation (pyruvate > glyoxylate > acetyl-CoA) and inactivation (glyoxylate > pyruvate > $$ " align="middle" border="0"> acetyl-CoA). The results showed that there is no direct relation between the extent of aggregation and the loss of enzymic activity.     

Low-resolution structure of the proteolytic fragments of the Rapana venosa hemocyanin in solution

Rapana venosa hemocyanin (Hc) is a giant oxygen-binding protein consisting of different subunits assembled in a hollow cylinder. The polypeptide chain of each subunit is believed to be folded in several oxygen-binding functional units of molecular mass 50 kDa, each containing a binuclear copper active site. Limited proteolysis with alpha-chymotrypsin of native R. venosa hemocyanin allows the separation of three functional proteolytic fragments with molecular masses of approximately 150, 100, and 50 kDa. The functional fragments, purified by combining gel filtration chromatography and ion-exchange FPLC, were analyzed by means of small-angle X-ray scattering (SAXS). The gyration radius of the 50-kDa Rapana Hc fraction (2.4 nm) agrees well with that calculated on the basis of the dimensions determined by X-ray crystallography for one functional unit of Octopus Hc (2.1 nm). Independent shape determination of the 50- and 100-kDa proteolytic fragments yields consistent low-resolution models. Simultaneous fitting of the SAXS data from these fragments provides a higher-resolution model of the 100-kDa species made of two functional units tilted with respect to each other. The model of the 150-kDa proteolytic fragment consistent with the SAXS data displays a linear chain-like aggregation of the 50-kDa functional units. These observations provide valuable information for the reconstruction of the three-dimensional structure of the minimal functional subunit of gastropod hemocyanin in solution. Furthermore, the spatial relationships among the different functional units within the subunit will help in elucidation of the overall quaternary structure of the oligomeric native protein.     

Structure of bacterial extracellular polymeric substances at different pH values as determined by SAXS

Extracellular polymeric substances (EPS) play an important role in cell aggregation, cell adhesion, and biofilm formation, and protect cells from a hostile environment. The EPS was isolated by trichloroacetic acid/ethanol extraction from broth culture of a marine bacterium isolate. The EPS was composed of glucose and galactose as determined by HPLC and TLC; the protein content was on average 15 ± 5% of EPS dry mass. The solution structure of EPS at different values of pH was revealed by small-angle x-ray scattering. Scattering curves of EPS solutions (0.4%, w/v) consistently showed two nearly linear log-log regions with slopes a and b in the q-ranges from 0.06 nm⁻¹ to 0.26 nm⁻¹, and from 0.27 nm⁻¹ to 0.88 nm⁻¹, respectively. Slope a was sensitive to pH changes whereas slope b was not. The observed sensitivity to pH was not a consequence of ionic strength variation with pH, as checked by salt addition. The pH variation causes major rearrangements of EPS structure mainly at length scales above 24 nm. To get a better understanding of the pH effect on EPS structure, the original model proposed by Geissler was refined into a mathematical model that enabled fitting of the experimental scattering curves in the pH range from 0.7 to 11.0. The model describes EPS structure as a network of randomly coiled polymeric chains with denser domains of polymeric chains. The results obtained from the model indicate that dense domains increase in average size from 19 nm at pH 11.0 to 52 nm at pH 0.7. The average distance between the polysaccharide chains at pH 0.7 was 2.3 nm, which indicates a compact EPS structure. Swelling was found to be at a maximum around pH = 8.8, where the average distance between the chains was 4.8 nm.     

The voltage-dependent step of the chloride transporter of Valonia utricularis encounters a Nernst-Planck and not an Eyring type of potential energy barrier

Voltage-clamp experiments were performed on cells of the giant marine alga Valonia utricularis to study the voltage dependence of the previously postulated chloride transporter (Wang, J., G. Wehner, R. Benz, and U. Zimmermann. 1991. Biophys. J. 59:235-248). Only one exponential current relaxation (apart from the capacitive spike) could be resolved up to a clamp voltage of ~120 mV within the time resolution of our experimental instrumentation (100 μs). This means that the rate constants of the heterogeneous complexation, k_R (association) and k_D (dissociation), were too fast to be resolved. Therefore, the “Läuger” model for carrier-mediated ion transport with equilibrium heterogeneous surface reaction was used to fit the experimental results. The voltage dependence of the initial membrane conductance was used for the evaluation of the voltage dependence of the translocation rate constant of the complexed carriers, k_AS. The initial conductance was found to be independent on the clamp voltage, which means that the translocation rate constant k_AS is a linear function of the applied voltage and that the voltage dependence of the translocation of charged carriers through the plasmalemma could be explained by a square-type Nernst-Planck barrier. The movement of the complexed form of the carrier through the membrane may be explained by a diffusion process rather than by simple first-order kinetic jump across an Eyring-type potential well. The current relaxation after a voltage clamp was studied as a function of the external chloride concentration. The results allowed an estimation of the stability constant, K, of the heterogeneous complexation reaction and a calculation of the translocation rate constants of the free and the complexed carriers, k_s and k_AS, respectively.     

Two Kinds of Protein Glycosylation in a Cell-Free Preparation from Developing Cotyledons of Phaseolus vulgaris

Membrane preparations from developing cotyledons of red kidney bean (Phaseolus vulgaris L.) transferred radioactive mannose from GDP-mannose (U-[¹⁴C]mannose) to endogenous acceptor proteins. The transfer was inhibited by the antibiotic tunicamycin, suggesting the involvement of lipidoligosaccharide intermediates typical of the pathway for glycosylation of asparagine residues. This was supported by the similarity of the linkage types of radioactive mannose in lipid-oligosaccharide and glycoprotein products; both contained labeled 2-linked mannose, 3,6-linked and terminal mannose typical of glycoprotein “core” oligosaccharides. As expected for “core” glycosylation, the transfer of labeled N-acetylglucosamine (GlcNAc) from UDP-GlcNAc (6-[³H]GLcNAc) to 4-linkage in endogenous glycoproteins could also be demonstrated. However, most of the radioactive GlcNAc was incorporated into terminal linkage, in a reaction insensitive to tunicamycin. The proteins receiving “core” oligosaccharide in vitro were heterogeneous in size, in contrast to those receiving most of the GlcNAc (which chiefly comprised the seed reserve-proteins phaseolin and phytohemagglutinin). It is suggested that following “core” glycosylation, single GlcNAc residues are attached terminally to the oligosaccharides of these seed proteins, without the involvement of lipid-linked intermediates. Phaseolin from mature seeds does not possess a significant amount of terminal GlcNAc and so it is possible that these residues are subsequently removed in a processing event.     

Single-Cell-Genomics-Facilitated Read Binning of Candidate Phylum EM19 Genomes from Geothermal Spring Metagenomes

The vast majority of microbial life remains uncatalogued due to the inability to cultivate these organisms in the laboratory. This “microbial dark matter” represents a substantial portion of the tree of life and of the populations that contribute to chemical cycling in many ecosystems. In this work, we leveraged an existing single-cell genomic data set representing the candidate bacterial phylum “Calescamantes” (EM19) to calibrate machine learning algorithms and define metagenomic bins directly from pyrosequencing reads derived from Great Boiling Spring in the U.S. Great Basin. Compared to other assembly-based methods, taxonomic binning with a read-based machine learning approach yielded final assemblies with the highest predicted genome completeness of any method tested. Read-first binning subsequently was used to extract Calescamantes bins from all metagenomes with abundant Calescamantes populations, including metagenomes from Octopus Spring and Bison Pool in Yellowstone National Park and Gongxiaoshe Spring in Yunnan Province, China. Metabolic reconstruction suggests that Calescamantes are heterotrophic, facultative anaerobes, which can utilize oxidized nitrogen sources as terminal electron acceptors for respiration in the absence of oxygen and use proteins as their primary carbon source. Despite their phylogenetic divergence, the geographically separate Calescamantes populations were highly similar in their predicted metabolic capabilities and core gene content, respiring O₂, or oxidized nitrogen species for energy conservation in distant but chemically similar hot springs.